Project Summary/Abstract Malaria remains one of the most devastating parasitic diseases in the world with the vast majority of deaths caused by Plasmodium falciparum. The pathology of the disease results exclusively from the blood-stage of the infection during which parasites invade and multiply within host erythrocytes. To establish this intracellular niche, P. falciparum imposes striking modifications to the erythrocyte through export of effector proteins but the export mechanism is poorly understood and the effector functions essential to parasite survival remain largely unknown. Export into the red blood cell requires crossing a vacuole membrane surrounding the parasite, a translocation event that depends upon the Plasmodium translocon of exported proteins (PTEX). In a recent paper, the candidate showed that inactivation of heat shock protein 101 (HSP101), a AAA+ ATPase component of PTEX, results in a complete block in protein export and parasite death. Related AAA+ proteins can unfold and directionally thread substrates through a central channel, suggesting HSP101 may drive recognition of effector proteins and power the translocation process. The proposed career development plan aims to dissect the role of HSP101/PTEX in export and to identify key exported effectors that enable the parasite to survive within the erythrocyte. Specific Aim 1 seeks to understand the substrate recognition and catalytic properties of recombinant HSP101 and to dissect the role of HSP101 in protein export within intact parasites. Specific Aim 2 will use genetic and proteomic approaches to define the function of additional PTEX components and a cross-linking mass spectrometry approach to map the architecture of the complex. Specific Aim 3 will analyze the function of exported effectors implicated in parasite survival within the erythrocyte using forward and reverse genetics as well as proteomic approaches. PTEX is an exciting new drug target and the proposed experiments will reveal key mechanistic information about the role of this export machinery as a necessary basis for rational drug design. Furthermore, identification of key exported proteins with roles in parasite survival may provide needed additional therapeutic targets. Collectively, this work will further our understanding of how the malaria parasite subverts its erythrocyte host cell and support development of new tools for control of malaria disease. The experience and research tools acquired in the process will propel the candidate into an established career as an independent investigator.